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Biomedical subjects

A Plückthun

Publications and source records attributed to A Plückthun.

At least 19 recordsLinked to original sources
PubMed2000

Viral escape at the molecular level explained by quantitative T-cell receptor/peptide/MHC interactions and the crystal structure of a peptide/MHC complex.

Viral escape, first characterized for the lymphocytic choriomeningitis virus (LCMV) in a mouse transgenic for the P14 T cell-receptor (TCR), can be due to mutations in T-cell epitopes. We have measured the affinity between the H-2D(b) containing the wild-type and two of its "viral escape" epitopes, as well as other altered peptide ligands (APL), by using BIACORE analysis, and solved the crystal structure of H-2D(b) in complex with the wild-type peptide at 2.75 A resolution. We show that viral escape is due to a 50 to 100-fold reduction in the level of affinity between the P14 TCR and the binary complexes of the MHC molecule with the different peptides. Structurally, one of the mutations alters a TCR contact residue, while the effect of the other on the binding of the TCR must be indirect through structural rearrangements. The former is a null ligand, while the latter still leads to some central tolerance. This work defines the structural and energetic threshold for viral escape.

Amino Acid Sequence↗
PubMed2000

The periplasmic Escherichia coli peptidylprolyl cis,trans-isomerase FkpA. II. Isomerase-independent chaperone activity in vitro.

We recently identified FkpA by selecting for the increased yield of antibody single-chain Fv (scFv) fragments in phage display, even of those not containing cis-prolines. We have now investigated the properties of FkpA in vitro. The peptidylprolyl cis-trans-isomerase activity of FkpA was found to be among the highest of any such enzyme with a protein substrate, yet FkpA is not able to enhance the proline-limited refolding rate of the disulfide-free hu4D5-8 scFv fragment, probably due to inaccessibility of Pro-L95. Nevertheless, the yield of the soluble and functional scFv fragment was dramatically increased in vitro in the presence of FkpA. Similar effects were observed for an scFv fragment devoid of cis-prolines. We are thus forced to conclude that the observed folding-assisting function is independent of the isomerase activity of the protein. The beneficial effect of FkpA was found to be due to two components. First, FkpA interacts with early folding intermediates, thus preventing their aggregation. Additionally, it has the ability to reactivate inactive protein, possibly also by binding to a partially unfolded species that may exist in equilibrium with the aggregated form, which may thus be released on a productive pathway. These in vitro measurements therefore fully reflect the in vivo results from periplasmic overexpression of FkpA.

Enzyme Stability↗
PubMed2000

Characterizing the functionality of recombinant T-cell receptors in vitro: a pMHC tetramer based approach.

The very low affinity of the T-cell receptor (TCR) for the peptide-major histocompatibility complex (pMHC) has made it very challenging to design assays for testing the functionality of these molecules on small scales, which in turn has severely hampered the progress in developing expression and refolding methodologies for the TCR. We have now developed an ELISA assay for detecting pMHC binding to functional recombinant TCRs. It uses tetramers of biotinylated pMHCs bound to a neutravidin-horseradish peroxidase conjugate and detects the presence of functional TCR, bound in a productive orientation to an immobilized anti-Cbeta antibody. Specificity can be stringently demonstrated by inhibition with monomeric pMHCs. The assay is very sensitive and specific, and requires only very small amounts of protein. It has allowed us to study the unstable recombinant TCR P14, which we expressed and refolded from Escherichia coli. The TCR P14 is directed against the most abundant epitope of LCMV. We have confirmed the specificity of the interaction by BIAcore, and were able to determine the dissociation constant of the interaction of the P14 TCR and of the gp33-pMHC as 6 microM. This affinity ranks it among the tighter ones of TCR-pMHC interactions, and unusually low affinity thus does not seem to be the cause of the modest protective power of these T-cells, compared to others elicited in the anti-LCMV response. This strategy of multimerizing one partner and immobilizing the other in both a native form and productive orientation should be generally useful for characterizing the weak interactions of cell-surface molecules.

Amino Acid Sequence↗
PubMed2000

Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides.

By analyzing the human antibody repertoire in terms of structure, amino acid sequence diversity and germline usage, we found that seven V(H) and seven V(L) (four Vkappa and three Vlambda) germline families cover more than 95 % of the human antibody diversity used. A consensus sequence was derived for each family and optimized for expression in Escherichia coli. In order to make all six complementarity determining regions (CDRs) accessible for diversification, the synthetic genes were designed to be modular and mutually compatible by introducing unique restriction endonuclease sites flanking the CDRs. Molecular modeling verified that all canonical classes were present. We could show that all master genes are expressed as soluble proteins in the periplasm of E. coli. A first set of antibody phage display libraries totalling 2x10(9) members was created after cloning the genes in all 49 combinations into a phagemid vector, itself devoid of the restriction sites in question. Diversity was created by replacing the V(H) and V(L) CDR3 regions of the master genes by CDR3 library cassettes, generated from mixed trinucleotides and biased towards natural human antibody CDR3 sequences. The sequencing of 257 members of the unselected libraries indicated that the frequency of correct and thus potentially functional sequences was 61 %. Selection experiments against many antigens yielded a diverse set of binders with high affinities. Due to the modular design of all master genes, either single binders or even pools of binders can now be rapidly optimized without knowledge of the particular sequence, using pre-built CDR cassette libraries. The small number of 49 master genes will allow future improvements to be incorporated quickly, and the separation of the frameworks may help in analyzing why nature has evolved these distinct subfamilies of antibody germline genes.

Amino Acid Sequence↗
PubMed2000

Correlation between in vitro stability and in vivo performance of anti-GCN4 intrabodies as cytoplasmic inhibitors.

A cellular assay system for measuring the activity of cytoplasmically expressed anti-GCN4 scFv fragments directed against the Gcn4p dimerization domain was established in the budding yeast Saccharomyces cerevisiae. The inhibitory potential of different constitutively expressed anti-GCN4 scFv intrabodies was monitored by measuring the activity of beta-galactosidase expressed from a GCN4-dependent reporter gene. The in vivo performance of these scFv intrabodies in specifically decreasing reporter gene activity was related to their in vitro stability, measured by denaturant-induced equilibrium unfolding. A framework-engineered stabilized version showed significantly improved activity, while a destabilized point mutant of the anti-GCN4 wild-type showed decreased effects in vivo. These results indicate that stability engineering can result in improved performance of scFv fragments as intrabodies. Increasing the thermodynamic stability appears to be an essential factor for improving the yield of functional scFv in the reducing environment of the cytoplasm, where the conserved intradomain disulfides of antibody fragments cannot form.

Amino Acid Sequence↗
PubMed2000

A heterodimeric coiled-coil peptide pair selected in vivo from a designed library-versus-library ensemble.

Novel heterodimeric coiled-coil pairs were selected simultaneously from two DNA libraries using an in vivo protein-fragment complementation assay with dihydrofolate reductase, and the best pair was biophysically characterized. We randomized the interface-flanking e and g positions to Gln, Glu, Arg or Lys, and the core a position to Asn or Val in both helices simultaneously, using trinucleotide codons in DNA synthesis. Selection cycles with three different stringencies yielded sets of coiled-coil pairs, of which 80 clones were statistically analyzed. Thereby, properties most crucial for successful heterodimerization could be distinguished from those mediating more subtle optimization. A strong bias towards an Asn pair in the core a position indicated selection for structural uniqueness, and a reduction of charge repulsions at the e/g positions indicated selection for stability. Increased stringency led to additional selection for heterospecificity by destabilizing the respective homodimers. Interestingly, the best heterodimers did not contain exclusively complementary charges. The dominant pair, WinZip-A1B1, proved to be at least as stable in vitro as naturally occurring coiled coils, and was shown to be dimeric and highly heterospecific with a K(D) of approximately 24 nM. As a result of having been selected in vivo it possesses all characteristics required for a general in vivo heterodimerization module. The combination of rational library design and in vivo selection presented here is a very powerful strategy for protein design, and it can reveal new structural relationships.

Amino Acid Sequence↗
PubMed2000

Novel fold and capsid-binding properties of the lambda-phage display platform protein gpD.

The crystal structure of gpD, the capsid-stabilizing protein of bacteriophage lambda, was solved at 1.1 A resolution. Data were obtained from twinned crystals in space group P21 and refined with anisotropic temperature factors to an R-factor of 0.098 (Rfree = 0. 132). GpD (109 residues) has a novel fold with an unusually low content of regular secondary structure. Noncrystallographic trimers with substantial intersubunit interfaces were observed. The C-termini are well ordered and located on one side of the trimer, relatively far from its three-fold axis. The N-termini are disordered up to Ser 15, which is close to the three-fold axis and on the same side as the C-termini. A density map of the icosahedral viral capsid at 15 A resolution, obtained by cryo-electron microscopy and image reconstruction, reveals gpD trimers, seemingly indistinguishable from the ones seen in the crystals, at all three-fold sites. The map further reveals that the side of the trimer that binds to the capsid is the side on which both termini reside. Despite this orientation of the gpD trimer, fusion proteins connected by linker peptides to either terminus bind to the capsid, allowing protein and peptide display.

Amino Acid Sequence↗
PubMed2000

Production of recombinant human beta2-microglobulin for scintigraphic diagnosis of amyloidosis in uremia and hemodialysis.

Amyloid of beta2-microglobulin (beta2m) origin can be diagnosed using 131I-radiolabelled-beta2m scintigraphy in patients with uremia and hemodialysis treatment. As the tracer beta2m is isolated from another patient, it carries the common risks, including viral infections such as Hepatitis B, C and HIV, which are associated with human plasma products. In order to exclude these risks we have produced recombinant human beta2m (rhbeta2m) in Escherichia coli. The expression vector pASK40DeltaLbeta2m(His)5 contains a C-terminal (His)5-tag for purification via immobilized metal ion affinity chromatography (IMAC). Size exclusion chromatography on a Superose 12 column represents the second step of purification. The isolated rhbeta2mH5 reacted in an immunochemically identical manner to native human beta2m, and showed a single band of approximately 11.8 kDa in Western blot analysis and revealed a single spot in two-dimensional gel electrophoresis. Mass spectrometry analysis revealed a single peak at the expected molecular mass of 12 415.8 Da. Uniformity was further proven by crystallization and N-terminal amino-acid sequence analysis. The rhbeta2mH5 protein was then produced under conditions that allow the intravenous use in humans. Intraveneously applied indium-111-labelled rhbeta2mH5 was monitored in hemodialysed patients with and without known beta2m-amyloidosis. The tracer was localized specifically to particular areas known to contain amyloid. Thus, this rhbeta2mH5 preparation is suitable for detecting amyloid-containing organs of the beta2m-class in vivo and fulfils the requirements of a tracer for common use. Finally, the use of indium-111 instead of iodine-131 has reduced the radioactive load and resulted in higher resolution.

Amyloidosis↗
PubMed2000

Direct evidence by H/D exchange and ESI-MS for transient unproductive domain interaction in the refolding of an antibody scFv fragment.

The refolding kinetics of a single-chain Fv (scFv) fragment, derived from a stabilized mutant of the phosphorylcholine binding antibody McPC603, was investigated by H/D exchange and ESI-MS and compared with the folding kinetics of its constituting domains V(H) and V(L). Both V(H) and V(L) adopt essentially native-like exchange protection within the dead time of the manual-mixing H/D exchange experiment (10 s) and in the case of V(L), which contains two cis-prolines in the native conformation, this fast protection is independent of proline cis/trans isomerization. At the earliest time point resolvable by manual mixing, fewer deuterons are protected in the scFv fragment than in the two isolated domains together, despite the fact that the scFv fragment is significantly more stable than V(L) and V(H). Full H/D exchange protection in the scFv fragment is gained on a time scale of minutes. This means that the domains in the scFv fragment do not refold independently. Rather, they associate prematurely and in nonnative form, a kinetic trap. Unproductive domain association is observed both after equilibrium- and short-term denaturation. For the equilibrium-denatured scFv fragment, whose native structure formation is dependent on a cis conformation of an interface proline in V(L), this cis/trans isomerization reaction proceeds about one order in magnitude more slowly than the escape from the trap to a conformation where full H/D exchange protection is already achieved. We interpret these data in terms of a general kinetic scheme involving intermediates with and without domain association.

Deuterium↗
PubMed1999

Selectively infective phage (SIP) technology: scope and limitations.

We review here the selectively infective phage (SIP) technology, a powerful tool for the rapid selection of protein-ligand and peptide-ligand pairs with very high affinities. SIP is highly suitable for discriminating between molecules with subtle stability and folding differences. We discuss the preferred types of applications for this technology and some pitfalls inherent in the in vivo SIP method that have become apparent in its application with highly randomized libraries, as well as some precautions that should be taken in successfully applying this technology.

Bacteriophage M13↗
PubMed1999

Ribosome display: an in vitro method for selection and evolution of antibodies from libraries.

Combinatorial approaches in biology require appropriate screening methods for very large libraries. The library size, however, is almost always limited by the initial transformation steps following its assembly and ligation, as other all screening methods use cells or phages and viruses derived from them. Ribosome display is the first method for screening and selection of functional proteins performed completely in vitro and thus circumventing many drawbacks of in vivo systems. We review here the principle and applications of ribosome display for generating high-affinity antibodies from complex libraries. In ribosome display, the physical link between genotype and phenotype is accomplished by a mRNA-ribosome-protein complex that is used for selection. As this complex is stable for several days under appropriate conditions, very stringent selections can be performed. Ribosome display allows protein evolution through a built-in diversification of the initial library during selection cycles. Thus, the initial library size no longer limits the sequence space sampled. By this method, scFv fragments of antibodies with affinities in the low picomolar range have been obtained. As all steps of ribosome display are carried out entirely in vitro, reaction conditions of individual steps can be tailored to the requirements of the protein species investigated and the objectives of the selection or evolution experiment.

Animals↗
PubMed1999

Domain interactions in antibody Fv and scFv fragments: effects on unfolding kinetics and equilibria.

The equilibrium denaturation and unfolding kinetics of the domains V(L) and V(H) have been compared with those of the Fv and single-chain Fv (scFv) fragment of an engineered variant of the antibody McPC603 in the presence and absence of the antigen phosphorylcholine. The scFv fragment is significantly more stable than the isolated constituting domains. Antigen binding stabilizes the heterodimeric assembly even further. Domain dissociation and domain unfolding are coupled processes, giving rise to a highly cooperative unfolding transition. For the Fv fragment, cooperative unfolding is only observed in the presence of antigen. At low protein concentrations and in the absence of antigen, the Fv fragment is significantly destabilized, leading to quantitative domain dissociation before significant domain unfolding takes place. The kinetic unfolding of V(H), V(L) and the scFv fragment is monophasic. Unfolding of the scFv fragment is much slower, when extrapolated to zero denaturant, than either of the isolated domains, suggesting that the higher thermodynamic stability of the scFv fragment is at least partially due to a high-energy transition state for unfolding. These studies emphasize the enormous importance of mutual domain stabilization in engineering stable antibodies.

Dose-Response Relationship, Drug↗
PubMed1999

Selection for improved protein stability by phage display.

A library of mutants of a single-chain Fv fragment (scFv) was generated by a combination of directed and random mutagenesis, using oligonucleotides randomized at defined positions and two rounds of DNA shuffling. The library was based on the already well folding and stable scFv fragment 4D5Flu. In order to further improve this framework and test the efficiency of various selection strategies, phage display selection was carried out under different selective pressures for higher thermodynamic stability. Incubation of the display phages at elevated temperatures was compared to exposure of the phages to high concentrations of guanidinium chloride. Temperature stress-guided selection yielded the most stable scFv mutant after two rounds of mutagenesis and selection, due to the irreversibility of the unfolding process. It possessed only two mutations (His(L27d)Asn and Phe(L55)Val) and showed a thermodynamic stability improved by roughly 4 kcal/mol, threefold better expression yields in Escherichia coli as well as a 20-fold better binding constant than the 4D5Flu wild-type. The selection results obtained in this study delineate the advantages, disadvantages and limitations of different stability stress selection methods in phage display.

Amino Acid Sequence↗
PubMed1999

High thermal stability is essential for tumor targeting of antibody fragments: engineering of a humanized anti-epithelial glycoprotein-2 (epithelial cell adhesion molecule) single-chain Fv fragment.

The epithelial glycoprotein-2 is abundantly expressed on many solid tumors and is a suitable target for antibody-based therapy. In the present study, an antiepithelial glycoprotein-2 single-chain Fv (scFv) was derived from the hybridoma MOC31 by phage display. Despite its high affinity (KD = 3.9 x 10(-9) M), however, this antibody fragment failed to significantly enrich at lung tumor xenografts in mice, mostly because of its insufficient thermal stability. To overcome this limitation, the antigen-binding residues of the MOC31 scFv fragment were grafted onto the framework of the highly stable and well-folding anti-c-erbB2 scFv 4D5. Further modification of the resulting 4D5 MOC-A, which was performed by transferring eight additional residues of the heavy chain variable domain core of the parent MOC31 antibody, produced 4D5 MOC-B, resulting in increased serum stability at 37 degrees C and also significantly improved expression behavior while retaining the antigen specificity and affinity of the parent MOC31 scFv. In mice, the scFv 4D5 MOC-B, which was radiolabeled with 99mtechnetium using a new histidine-tag specific labeling method (Waibel et al., Nature Biotechnol., 17: 897-901, 1999), showed favorable blood clearance and efficient enriches at lung tumor xenografts, with a tumor:blood ratio of 5.25 and a total dose of 1.47% injected dose per gram after 24 h. Biophysical properties such as high thermal stability are thus decisive for whether these molecules are useful in vivo, and our approach may provide a general strategy to solve this problem. This is also the first report of using a humanized anti-EGP-2 scFv in vivo for targeting solid tumors, which is a promising targeting moiety for the diagnostics and therapy of EGP-2-positive tumors in patients.

Amino Acid Sequence↗
PubMed1999

Circular beta-lactamase: stability enhancement by cyclizing the backbone.

We have cyclized the polypeptide backbone of beta-lactamase with a short peptide loop as a novel method for protein stabilization, using intein-mediated protein ligation. Successful cyclization was proven by mass spectrometry and subsequent re-linearization by proteolytic cleavage, as well as by resistance against carboxypeptidase. Under the conditions of the experiment, no disulfide bond is present. The circular form of beta-lactamase was found to be significantly more stable against irreversible aggregation upon heating than the linear form. The circular form could be purified from the linear one either by this heat treatment or by a his-tag which became exopeptidase-resistant by cyclization. The increased stability of the circular form is probably due to the decreased conformational entropy in the unfolded state and in the intermediate states. While the introduction of additional disulfide bonds for protein stabilization follows the same rationale, the cyclization strategy may disturb the structure less and thus constitute a general method for stabilizing those proteins with N- and C-termini in close proximity.

Amino Acid Sequence↗